Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues.

Axon pathfinding relies on cellular signaling mediated by growth cone receptor proteins responding to ligands, or guidance cues, in the environment. Eph proteins are a family of receptor tyrosine kinases that govern axon pathway development, including retinal axon projections to CNS targets. Recent examination of EphB mutant mice, however, has shown that axon pathfinding within the retina to the optic disc is dependent on EphB receptors, but independent of their kinase activity. Here we show a function for EphB1, B2 and B3 receptor extracellular domains (ECDs) in inhibiting mouse retinal axons when presented either as substratum-bound proteins or as soluble proteins directly applied to growth cones via micropipettes. In substratum choice assays, retinal axons tended to avoid EphB-ECDs, while time-lapse microscopy showed that exposure to soluble EphB-ECD led to growth cone collapse or other inhibitory responses. These results demonstrate that, in addition to the conventional role of Eph proteins signaling as receptors, EphB receptor ECDs can also function in the opposite role as guidance cues to alter axon behavior. Furthermore, the data support a model in which dorsal retinal ganglion cell axons heading to the optic disc encounter a gradient of inhibitory EphB proteins which helps maintain tight axon fasciculation and prevents aberrant axon growth into ventral retina. In conclusion, development of neuronal connectivity may involve the combined activity of Eph proteins serving as guidance receptors and as axon guidance cues.

Diffuse uterine leiomyomatosis with ovarian and parametrial involvement.

BACKGROUND: Diffuse uterine leiomyomatosis is a rare, benign entity and approximately 30 cases have been described previously. CASE: A 42-year-old woman who complained of abdominal pain had a pelvic ultrasound scan showing a uterine mass. During the operation, the surgeon observed that both ovaries, the broad ligament, and the pelvis contained various nodules of striking size. On sectioning, uterus and ovaries contained multiple nodules of elastic consistency; microscopically, all consisted of benign smooth muscle tissue. CONCLUSION: Leiomyomatosis may exhibit concomitant parametrial, pelvic, and bilateral ovarian involvement.

Cambio de tipo de células de Paneth en el adenocacinoma de próstata: a propósito de un caso / Change in type of Paneth´s granular cells in adenocarcinoma of the prostate: A case report

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Gallbladder motility and lithogenesis in obese patients during diet-induced weight loss.

UNLABELLED: Obesity and weight loss are important risk factors for gallstone development. The mechanisms involved are unknown. We prospectively studied changes in gallbladder (GB) emptying and bile composition during weight loss. We studied 12 alithiasic obese subjects who entered a six-month diet program (800-1200 kcal/day, 26 g fat/day). As controls we evaluated 12 healthy nulliparous nonobese young women. GB volumes were studied by ultrasonography (fasting volume, GBFV; residual volume after a liquid meal, GBRV) at entry and after 4 and 20 weeks of dieting. Bile acid pool size, biliary lipid composition, presence of cholesterol crystals, and nucleation time were also studied. Of 12 obese subjects studied (mean BMI 35.1 kg/m2), 10 remained in the program for six months, but only six completed the entire study protocol, obtaining a significant weight loss (BMI: 31.2 kg/m2, P < 0.001). GBFV was greater in obese subjects than in nonobese controls (27.5 +/- 10.7 vs 11.7 +/- 6 ml; P < 0.05). GBRV and GB emptying curves were similar in both groups and did not change during weight loss. The obese subject who developed gallstones (1/10) was the only one who had cholesterol crystals in bile and a sluggish initial GB emptying. IN CONCLUSION: (1) obese subjects had a greater GBFV than controls; however, the GB emptying was adequate. (2) During weight loss we did not observe significant changes in GB kinetics or the bile parameters studied. (3) We observed a relatively low frequency of gallstone formation, which can be explained by a high fat content of the diet (26 g/day) and by the adequate GB emptying of our group of patients. (4) An abnormal GB contractility and cholesterol crystals in bile could be considered premonitory to gallstone formation.

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation.

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.

Brain-derived TNFalpha mediates neuropathic pain.

Neuropathic pain is a chronic pain state that develops a central component following acute nerve injury. However, the pathogenic mechanisms involved in the expression of this central component are not completely understood. We have investigated the role of brain-associated TNF in the evolution of hyperalgesia in the chronic constriction injury (CCI) model of neuropathic pain. Thermal nociceptive threshold has been assessed in rats (male, Sprague-Dawley) that have undergone loose, chromic gut ligature placement around the sciatic nerve. Total levels of TNF in regions of the brain, spinal cord and plasma have been assayed (WEHI-13VAR bioassay). Bioactive TNF levels are elevated in the hippocampus. During the period of injury, hippocampal noradrenergic neurotransmission demonstrates a decrease in stimulated norepinephrine (NE) release, concomitant with elevated hippocampal TNF levels. Continuous intracerebroventricular (i.c.v.) microinfusion of TNF-antibodies (Abs) starting at four days, but not six days, following ligature placement completely abolishes the hyperalgesic response characteristic of this model, as assessed by the 58 degrees C hot-plate test. Antibody infusion does not decrease spinal cord or plasma levels of TNF. Continuous i.c.v. microinfusion of rrTNF alpha exacerbates the hyperalgesic response by ligatured animals, and induces a hyperalgesic response in animals not receiving ligatures. Likewise, field-stimulated hippocampal adrenergic neurotransmission is decreased upon continuous i.c.v. microinfusion of TNF. These results indicate an important role of brain-derived TNF, both in the pathology of neuropathic pain, as well as in fundamental pain perception.

Serotonergic binding in the rat dorsal raphe nucleus: critical role of MAO inhibition.

The biochemical properties of the 5-HT1A receptor in dorsal raphe nucleus (DRN) were investigated using a micropunch procedure. Initially, the Ki value for 5-hydroxytryptamine (5-HT) binding to a site labeled by the 5-HT1A-selective ligand [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was 20-fold higher than the KD for [3H]5-HT. In addition, a number of putative 5-HT1A selective ligands displayed poor affinity for the [3H]8-OH-DPAT site. The possibility that these discrepant results were due to metabolism of the receptor ligands was investigated by increasing the concentration of the monoamine oxidase (MAO) inhibitor, pargyline. Increasing the concentration of pargyline reduced, but did not abolish, the discrepancy between the Ki and KD values for 5-HT. However, inclusion of clorgyline, which is a more potent MAO inhibitor, resulted in an-excellent agreement between the Ki and KD values for 5-HT. In addition, when clorgyline was used, 5-HT1A-selective compounds displayed high affinity for the DRN binding site consistent with [3H]8-OH-DPAT labeling a 5-HT1A receptor in this tissue. The present study describes a fast and easy method for measuring biochemical properties in small discrete brain areas. These studies also indicate that pargyline should be replaced in serotonergic binding assays with a more potent inhibitor of monoamine oxidase such as clorgyline.

The effects of a pulsed Nd:YAG laser on subgingival bacterial flora and on cementum: an in vivo study.

The purpose of this study was to compare the effects of scaling and Nd:YAG laser treatments with that of scaling alone on cementum and levels of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, and Treponema denticola. Study samples consisted of 14 patients, age 30 to 75 years, 8 females and 6 males, with a total of 150 periodontally involved sites with probing depth > or = 5 mm. Group A consisted of 100 pockets that were subdivided into 4 equal groups that were treated with conventional scaling and pulsed Nd:YAG laser using an optic fiber of 300 microns and 4 different power levels as follows: Group 1: P = 0.8 W, f = 10 Hz, E = 100 mJ/pulse; Group 2: P = 1.0 W, f = 1.0 Hz, E = 100 mJ/pulse; Group 3: P = 1.2 W, f = 12 Hz, E = 100 mJ/purse; and Group 4: P = 1.5 W, f = 15 Hz, E = 100 mJ/pulse. The time of each treatment was 60 sec per pocket in all 4 groups. Group B consisted of 50 pockets that were treated by conventional scaling alone and served as a control group. Microbiological samples from group A were collected before scaling; after scaling = before laser, just after laser, 2 weeks later, 6 weeks later, and 10 weeks later. Microbiological samples from group B were collected before scaling, after scaling, 6 weeks later, and 10 weeks later. Microbiological analysis of all samples was done by the Institute Für Angewandte Immunologie (IAI) method. The effects of laser on root surfaces were assessed by SEM examination and the sample consisted of 13 teeth from 5 different patients. Four sets of 3 teeth each were treated with Nd:YAG laser using 0.8, 1.0, 1.2, and 1.5 W, respectively. One tooth was just scaled and not treated with laser to serve as a control. Microbiological analysis of Group A samples indicated posttreatment reduction in levels of all 4 bacterial types tested compared to pretreatment levels and Group B controls. SEM examination of the specimens treated with Nd:YAG laser at different levels exhibited different features of root surface alterations.

[Study of volume and kinetics of gallbladder emptying in non-pregnant, pregnant, and puerperal women]. / Estudio del volumen y de la cinética del vaciamiento vesicular en mujeres nuligestas, en embarazadas y puérperas.

Gallbladder stasis apparently plays an important role in the patho-genesis of cholelithiasis during pregnancy. On the other hand, gallstones diagnosed immediately after delivery disappear spontaneously during late puerperium in one third of these patients. Gallbladder emptying was assessed by biliary scintigraphy and ultrasonography in normal, nulliparous volunteers. The two methods had an excellent correlation: thence, we used ultrasonography to determine gallbladder volume and contraction in pregnant and puerperal women. Fasting and postprandial residual volumes were significantly larger during pregnancy, while the kinetics f gallbladder emptying was similar in nulliparous and pregnant women. During puerperium, gallbladder volume returned to the values observed in nulliparae; but the kinetics of emptying was significantly faster, suggesting an increased sensitivity of gallbladder muscle to physiologic stimuli.

Effect of high-energy phosphates and free radical scavengers on replant survival in an ischemic extremity model.

In replantation surgery, preoperative and intraoperative ischemia can lead to irreversible changes that prevent reperfusion during the subsequent re-establishment of circulation. These changes are termed the no-reflow phenomenon. Ischemic phase damage was addressed by comparing the dose-response effects of controls vs. five different high-energy phosphate compounds on replanted limb survival. Reperfusion damage was evaluated via comparisons of controls with superoxide dismutase (SOD). Ischemic hindlimbs treated with high-energy phosphates displayed improved survival compared with controls. Limbs treated with SOD demonstrated no change in survival at 4 hours and improved survival at 8 hours. Combining adenosine and SOD had no improved effect on survival. Adenosine was the most effective high-energy phosphate in limiting ischemic damage. The free radical scavenger (SOD) was beneficial only at the later stages of ischemia. In this experimental model, there appears to be a role for both phosphates and free radical scavengers in enhancing ischemic tissue survival.

Role of G-proteins in ciliary process adenylyl cyclase responses of the albino rabbit eye.

After intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5 -1 microgram/eye) into the albino rabbit eye, the in vitro responses of ciliary process adenylyl cyclase (AC) to isoproterenol, vasoactive intestinal peptide (VIP), and forskolin (FSK) were increased 21-40% for PTX, but for CTX-injected eyes AC responses to fluoroaluminate, VIP and FSK decreased 70-50%. The increased responses after PTX suggests that this toxin blocked an inhibitory Gi control of AC that is present in the control tissue. However, prolonged (> 24 hr) in vivo exposure to CTX appears to down-regulate the AC enzyme. In contrast to the in vivo findings, AC responsiveness was unaffected by PTX pre-treatment of membranes in vitro, while CTX pre-treatment increased basal activity (+600%), and the FSK response (+30%), but decreased responsiveness to fluoroaluminate, VIP and isoproterenol by 88-56%. Treatment of ciliary process membranes with 32P-NAD and CTX or PTX followed by SDS-PAGE autoradiography of labelled proteins gave two bands for the G-protein alpha-subunits of Gs (45, 56 kDa) and one broad band centered at 40 kDa for Gi-type subunits respectively. Western blots using specific antibodies showed the presence of Gi type I or III, but no detectable Gi type II or Go in rabbit ciliary processes. We conclude that the changes in adenylyl cyclase enzyme responses after intraocular CTX or PTX may not correlate with cAMP levels and intraocular pressure effects. However, the in vitro biochemical data on AC responses and on G-proteins provide evidence for dual regulation of ciliary process AC by activating and inhibitory G-proteins.

Endotoxins in cholera and pertussis toxins interfere with in vivo responses to these agents in the albino rabbit eye.

Intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5-1 microgram/eye) decreased intraocular pressure (IOP) up to 50% in the albino rabbit eye, which lasted up to six days. Both toxins were active on G-proteins as determined by in vitro and in vivo effects on ciliary process adenylyl cyclase activity and by ADP ribosylation of G-protein alpha-subunits with 32P-NAD. However, forty-two hours after toxin injection aqueous humor proteins increased from control levels of 0.8-1.2 mg/ml to 8-25 mg/ml. Both toxins contained 1-3 parts per thousand endotoxin sufficient to cause the IOP and aqueous humor protein responses observed. We conclude that the in vivo responses to intraocular CTX or PTX obtained from commercial sources may not provide unequivocal evidence for the role(s) of G-proteins in aqueous humor dynamics, and must be interpreted with caution.

Effects of Al3+ and Be2+ ions combined with NaF on ciliary process adenylyl cyclase activity and aqueous humor dynamics in the rabbit eye.

PURPOSE: The activity of Al3+, Ga3+, and Be2+ ions in the presence of NaF to directly activate G-proteins was investigated by their potentiative effect on forskolin (FSK)-activated adenylyl cyclase in rabbit ciliary process membranes and their effects on aqueous humor dynamics in vivo. METHODS: Adenylyl cyclase (AC) was determined by radiometric conversion of ATP to cAMP by the particulate fraction of rabbit ciliary processes. Intravitreal injections of sterile solutions of analytical grade salts were made into the center of the vitreous in a volume of 20 microliters. Intraocular pressure, aqueous humor flow, and uveoscleral outflow measurements were made by pneumatonometry, fluorophotometry, and fluorescein-dextran method, respectively. Outflow facility was determined by tonography in the intact eyes and by two-level constant pressure perfusion in cannulated eyes. RESULTS: Both Al3+ (EC50, 40 mumol/l) and Be2+ (EC50, 11 mumol/l) in the presence of 0.5-2 mM NaF activated the stimulatory G-protein Gs. Ga3+ was ineffective and did not antagonize the activation by Al3+. Intravitreal injections of Al3+ (1 mumol/eye) or Be2+ (0.5 or 1 mumol/eye) had no significant intraocular pressure (IOP) effect, nor did 1.5 or 3 mumol/eye of NaF, but when either cation was injected together with NaF, IOP decreased by up to 40% for up to 140 hr. At the time of maximum IOP effect (72 hr) aqueous humor flow determined by fluorophotometry was decreased in BeCl2+ NaF-treated eyes by 40% relative to BeCl2-treated eyes; however, tonographic facility of outflow was unaffected. Uveoscleral flow was also decreased by 38% in BeCl2+ NaF treated eyes. CONCLUSIONS: These findings support the hypothesis that Gs activation of ciliary process adenylyl cyclase decreases aqueous humor formation rate in rabbit eyes, and that activation of G-proteins mediates contraction of ciliary muscles causing a decrease of aqueous humor outflow via the uveoscleral route. The results suggest that G-proteins putatively involved in trabecular facility changes are less sensitive to activation by BeF3- than are other parameters of aqueous humor dynamics.

Co-cultures of microglia and astrocytes from kainic acid-lesioned adult rat hippocampus: effects of glutamate.

The long-standing question concerning the direct actions of glutamate on the membrane potential of astroglial cells in the central nervous system was addressed using the in vitro kainic acid-lesioned hippocampal slice preparation and primary cell co-cultures of astrocytes and microglia derived from such lesions. The ultrastructure of the lesioned hippocampus was examined to aid in the identification of the cells appearing in culture. In culture, microglia appeared as flat cells, less than 1 micron in thickness at the edge of the cell, but thicker (about 5 microns) near the nucleus. The cytoplasm was packed with granular inclusions. Microglia appeared in two morphological forms, amoeboid and ramified. The amoeboid form was characterized by a cell body with a single process, and was always observed 1 day after starting the cell culture. Such cells became less frequent after 1 week in culture. The ramified form appeared as a rounded cell, devoid of processes, and were frequently observed in older cultures (greater than 1 week). Microglia did not round up after exposure to dibutyrylcyclic adenosine monophosphate (cAMP), and did not stain for glial fibrillary acidic protein (GFAP). An ultrastructural examination of the lesion demonstrated that microglia were present and that they contained many cytoplasmic granules similar to lipofuscin-containing granules. No filaments were observed in the cytoplasm of microglia. By contrast, the cytoplasm of astrocytes in culture had far fewer granules, rounded up to dibutyryl-cAMP, exhibited multiple processes, and stained for GFAP. In slices, astrocytes had no lipofuscin-containing granules, but numerous cytoplasmic filaments were present.(ABSTRACT TRUNCATED AT 250 WORDS)

MR imaging of normal rat brain at 0.35 T and correlated histology.

A custom-built small-animal transceiver was used for in vivo imaging of normal rat brain at 0.35 T, with the objective of identifying anatomic components by comparison of images with corresponding histologic sections. The cerebrum, cerebellum, brain stem, ventricles, hippocampus, and subarachnoid space were identified and cerebrospinal fluid (CSF) was differentiated from gray matter and white matter on coronal and transaxial magnetic resonance (MR) images. These images compare favorably with those obtained by others at higher field strengths in regard to delineating major neuroanatomic structures. It is concluded that this technique will be useful for investigating small-animal models of human neurologic disease involving morphologic and morphometric changes in gray matter, white matter, and CSF-filled spaces.

Morphometry of the lumbar spine: anatomical perspectives related to transpedicular fixation.

The pedicles of lumbar vertebrae were measured both directly and radiographically to determine the differences between the sexes and the accuracy of radiographic measurement. The lumbar pedicles of cadavera of forty-nine patients--twenty-four men and twenty-five women--who died between the ages of sixty and ninety-eight years were measured directly and on radiographs. The pedicles of lumbar vertebrae from fifty-one patients--twenty-three men and twenty-eight women--between the ages of twenty and fifty years who had low-back problems were measured on radiographs and computerized tomographic scans. Comparison revealed that the average transverse and sagittal diameters of the pedicles and the distance from the posterior aspect of the laminar cortex to the anterior aspect of the cortex of the vertebral body along the central axis of the pedicles were 5 to 20 per cent greater in men, but the transverse and sagittal angles of the pedicle did not differ significantly between the sexes. Measurements on radiographs and computerized tomographic scans of the transverse angles of the pedicles and of the distances from the posterior aspect of the laminar cortex to the anterior aspect of the cortex of the vertebral body from the second to the fifth lumbar vertebra were greater than direct measurements, even without magnification. Direct measurements of the diameters of the transverse and sagittal diameters of the pedicle of the fifth lumbar vertebra, however, were greater than the radiographic measurements.

[Behavior of a dental rotary instrument alloy after titanium nitride coating]. / Etude du comportement de l'alliage des instruments rotatifs dentaires après revêtement de nitrure de titane.

A titanium nitride coating has been deposited on steel and tungsten carbide dental rotary instruments. This process is carried out under partial vacuum, by reacting vapourized metal (titanium) and nitrogen in a plasma. The instruments temperature is around 500 degrees C during coating. We have determined by micro-analysis the treatment modifications upon the different bursa components. VICKERS microhardness tests have shown the coating temperature effects towards the heat treatments of employed steels.